We provide three lines of evidence that at least part of the machinery of PCD is involved in lens fiber differentiation. First, we show that the nucleus in developing secondary lens fiber cells can be labeled by the TUNEL technique, confirming the findings of Bassnett and Mataic (1997)
, who demonstrated DNA fragmentation in differentiating chick lens fibers by both the TUNEL technique and in situ electrophoresis. Together with the earlier findings of Modak and colleagues (Modak and Perdue, 1970
; Appleby and Modak, 1977
), these results indicate that DNA fragmentation, a characteristic feature of PCD, occurs as lens cells differentiate and lose their nucleus. It is uncertain why some previous studies failed to find TUNEL labeling of secondary lens fiber cell nuclei in the developing mouse lens (Fromm et al., 1994
; Morgenbesser et al., 1994
; Pan and Griep, 1994
; Chow et al., 1995b
; Robinson et al., 1995
). It is possible, as Bassnett and Mataic (1997)
suggest, that the discrepancies in TUNEL labeling reflect differences in the way the labeling procedures were carried out. No labeling has been observed, for example, where the sections were not treated with proteinase K before incubation with TdT, while nonspecific labeling has been prominent when streptavidin– fluorescein was used without pretreatment with BSA.
The second line of evidence that components of the cell death program are involved in lens fiber differentiation is that PARP is cleaved in the developing lens. PARP cleavage is a widely used indicator of both PCD and the activation of caspase-3 and/or one or more of its close relatives such as caspase-7 (Alnemri et al., 1996
). PARP is cleaved from a 116-kD form to an 85-kD fragment in many examples of PCD (Kaufmann, 1989
; Kaufmann et al., 1993
; Gu et al., 1995
; Nicholson et al., 1995
; Tewari et al., 1995
), and it is cleaved in a similar way in the developing lens. This finding strongly suggests that caspase-3, and/or a closely related caspase, is activated during lens fiber differentiation, which in turn suggests that some parts of the machinery of PCD are used in normal lens development. It seems likely, for example, that caspase-6 becomes activated and cleaves one or more of the nuclear lamins (Fernandes- Alnemri et al., 1995a
; Orth et al., 1996
; Takahashi et al., 1996
) to help remove the nuclear lamina when the nucleus degenerates in developing lens fibers.
The third line of evidence that lens fiber differentiation involves the machinery of PCD is that the caspase inhibitor zVAD-fmk, but not the chemically similar cathepsin B inhibitor zFA-fmk, inhibits both the formation of anucleate lentoid bodies and PARP cleavage in explant cultures of lens epithelial cells treated with bFGF and insulin. In this in vitro model of lens fiber differentiation, the production of β-crystallin increases, just as in vivo (Peek et al., 1992
), and it is interesting that this increase is not suppressed by zVAD-fmk treatment. These findings suggest that, whereas PARP cleavage and the loss of the nucleus during lens fiber differentiation requires activated caspases, this is not the case for some other aspects of lens fiber differentiation, such as the increased expression of β-crystallin. Thus, lens fiber differentiation is not simply PCD, since the outcome is very different from that of classical PCD, where the cell usually shrinks, frequently fragments, and is rapidly phagocytosed and digested (Kerr et al., 1972
; Wyllie et al., 1980
); a lens fiber, by contrast, elongates, fills up with crystallins, and persists for the lifetime of the animal.
Taken together, our results provide strong evidence that the differentiation of a lens epithelial cell into a lens fiber involves components of the basic machinery of PCD. It was recently reported that mice in which the caspase-3 gene was deleted by targeted gene disruption die perinatally with an excess of cells in the central nervous system, apparently as a result of decreased PCD in neuroepithelial cells (Kuida et al., 1996
). Although PCD was reported to occur normally in other organs, it appears to us that there is an abnormally large number of nuclei in the core of the lens of the caspase-3–deficient mouse shown in Fig. in Kuida et al. (1996)
, which could reflect a failure of fiber cell denucleation. If so, this finding would provide further evidence that lens fiber differentiation requires components of the basic machinery of PCD.
In addition to lens cell differentiation, there are at least two other cases where mammalian cell differentiation is accompanied by the loss of the nucleus and other organelles—the development of erythrocytes and skin keratinocytes. It will be interesting to see if the differentiation in these cases is also caspase dependent. This seems likely in the case of keratinocytes, as overexpression of the PCD-suppressing gene bcl-2
has been reported to inhibit human keratinocyte differentiation in culture (Nataraj et al., 1994